1. Field of the Invention
The present invention relates generally to the field of cancer diagnostics. More particularly, it concerns markers for use in the diagnosis and prognosis of cancer. Also provided are related protein, DNA and antibody compositions and various methods of use thereof, including methods for cancer diagnosis and treatment.
2. Description of Related Art
Cancer is one of the leading causes of disease, being responsible for 526,000 deaths in the United States each year (Boring et al., 1993). For example, breast cancer is the most common form of malignant disease among women in Western countries and, in the United States, is the most common cause of death among women between 40 and 55 years of age (Forrest, 1990). The incidence of breast cancer is increasing, especially in older women, but the cause of this increase is unknown. Malignant melanoma is another form of cancer whose incidence is increasing at a frightening rate, at least sixfold in the United States since 1945, and is the single most deadly of all skin diseases (Fitzpatrick, 1986).
One of the most devastating aspects of cancer is the propensity of cells from malignant neoplasms to disseminate from their primary site to distant organs and develop into metastases. Despite advances in surgical treatment of primary neoplasms and aggressive therapies, most cancer patients die as a result of metastatic disease. Animal tests indicate that about 0.01% of circulating cancer cells from solid tumors establish successful metastatic colonies (Fidler, 1993).
Thus, the detection of occult cancer cells in circulation is important in assessing the level of tumor progression and metastasis. Because subclinical metastasis can remain dormant for many years, monitoring of patients"" blood for circulating tumor cells may prove advantageous in detecting tumor progression before metastasis to other organs occurs. Assessment of circulating tumor cells also would provide a rapid monitoring system to determine if a specific therapy is effective.
The recognition or lack of recognition of cancer cells by a host organism is a complicated process. Understanding of the field presumes some understanding of both basic immunology and oncology. Early research on mouse tumors revealed that these cells displayed molecules which led to rejection of tumor cells when transplanted into syngeneic animals. These molecules are xe2x80x9crecognizedxe2x80x9d by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was obtained with tumors induced in vitro by chemical carcinogens (Prehn, et al., 1957; Klein et al., 1960; Gross, 1943, Basombrio, 1970), as well as on tumors induced in vitro via ultraviolet radiation. (Kripke, 1974). The antigens expressed by the tumors and which elicited the T-cell response were found to be different for each tumor. This class of antigens has come to be known as xe2x80x9ctumor specific transplantation antigensxe2x80x9d (TSTAs).
While T-cell mediated immune responses were observed for tumors induced through the application of carcinogens, spontaneous tumors were thought to be generally non-immunogenic. These were, therefore, believed not to present antigens which provoked a response to the tumor in the tumor carrying subject (Hewitt, et al., 1976).
Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor (Van Pel et al., 1983). Thus, it has been shown that it is possible to elicit presentation of a so-called xe2x80x9ctumor rejection antigenxe2x80x9d in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors (Fearon et al., 1988) in this regard.
A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytolytic T cells, leading to lysis. This class of antigens will be referred to as xe2x80x9ctumor rejection antigensxe2x80x9d (TRAs). TRAs may or may not elicit antibody responses. These antigens have been studied by cytolytic T cell characterization studies, i.e., the in vitro study of the identification of the antigen by a particular cytolytic T cell (CTL) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed. Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. (Levy et al., 1977; Boon et al., 1980; Brunner et al., 1980; Maryanski et al., 1980; Maryanski et al., 1982; Palladino et al., 1987).
U.S. Pat. No. 5,342,774, incorporated herein by reference, described a family of human tumor rejection antigen precursor coding genes, referred to as the MAGE family (van der Bruggen et al., 1991; Traversari et al., 1992). It now is clear that the various genes of the MAGE family are expressed in tumor cells, and can serve as markers for the diagnosis of such tumors, as well as for other purposes discussed therein.
Although MAGE expression has been identified in different types of cell lines and tumor tissues, it is not ubiquitously expressed in tumors of all types. Similar molecules, such as PAGE, BAGE (Boel et al., 1995) and GAGE (van den Eynde, 1995) have been identified as tumor recognition antigens for a variety of cancers including melanoma, sarcomas, non-small cell lung cancers, head and neck tumors, bladder tumors and prostate tumors. However, there remains a significant scientific hurdle in identifying which MAGE gene will be expressed by a particular tumor type. Thus, while on one level one can say that MAGE, BAGE and PAGE genes are xe2x80x9cmarkersxe2x80x9d for tumors, on the level of specific tumor types, the correlation of marker and tumor type is not predictable, and must be determined empirically. Thus, there is a need to define tumor marker antigens that are expressed ubiquitously on all cancer types.
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing nucleic acid species and marker genes that are expressed in cancers cells, and methods of making and using such nucleic acids and related proteins and antibodies. Further provided are methods for the diagnosis, prognosis and treatment of cancers using one or more of the foregoing compositions.
Thus, in order to meet the objectives of the present invention, there is provided herein a DNA segment comprising an isolated gene that encodes a HOJ-1 polypeptide. In particular embodiments, there is provided a gene that encodes a human HOJ-1 polypeptide. In other preferred embodiments, there is provided a gene that encodes a HOJ-1 protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:2. In still other embodiments, the gene includes a contiguous nucleic acid sequence from SEQ ID NO:1. In certain embodiments, the present invention provides a nucleic acid segment that encodes a HOJ-1 peptide of from about 105 to 109 amino acids in length.
The DNA segment may comprise a gene that encodes the HOJ-1 protein of SEQ ID NO:2. In other embodiments, the present invention comprises a gene that has the nucleic acid sequence of SEQ ID NO:1. In particular aspects, the gene is positioned under the control of a promoter. In preferred aspects, the gene is positioned under the control of a recombinant promoter in an expression vector. In other specific embodiments, the gene is positioned in reverse orientation under the control of a promoter, the promoter expressing an antisense product. In particular embodiments, the DNA segment may be further defined as a recombinant vector.
Other aspects of the present invention provide a recombinant host cell comprising a DNA segment that comprises an isolated gene that encodes a HOJ-1 protein. In preferred embodiments the recombinant host cell of the present invention further may be defined as a prokaryotic host cell. In other embodiments, the recombinant host cell of the present invention further may be defined as a eukaryotic host cell. In particular aspects, the DNA segment is introduced into the cell by means of a recombinant vector. In preferred embodiments, the host cell expresses the DNA segment to produce the encoded HOJ-1 protein or peptide. In more preferred embodiments, the expressed HOJ-1 protein or peptide includes a contiguous amino acid sequence from SEQ ID NO:2.
The present invention also contemplates a method of using a DNA segment that encodes a HOJ-1 protein or peptide, comprising expressing the DNA segment in a recombinant host cell and collecting the HOJ-1 protein or peptide expressed by the cell.
In further aspects, the present invention provides an isolated nucleic acid segment comprising a sequence region that consists of at least 14 contiguous nucleotides that have the same sequence as, or are complementary to, 14 contiguous nucleotides of SEQ ID NO:1.
In still further aspects, the present invention contemplates an isolated nucleic acid segment of from 14 to about 888 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1, or a complement thereof, under standard hybridization conditions. In other embodiments, there is provided an isolated nucleic acid segment of from 14 to about 888 nucleotides in length that hybridizes under high stringency conditions to the nucleic acid segment of SEQ ID NO:1, or a complement thereof. The nucleic acid segment further may be defined as a DNA segment. In preferred embodiments, the nucleic acid segment further may be defined as a RNA segment.
Also provided by the present invention is an isolated nucleic acid segment comprising a contiguous nucleic acid sequence that is identical or complementary to the nucleic acid sequence of SEQ ID NO:1. Another aspects of the present invention provides an isolated nucleic acid segment having the contiguous nucleic acid sequence of SEQ ID NO:1, or a complement thereof. In preferred embodiments, the nucleic acid may be selected from the group consisting of genomic DNA, complementary DNA and RNA. In particular embodiments, the nucleic acid is a complementary DNA and further comprises a promoter operably linked to the region, or the complement thereof, encoding a HOJ-1 tumor antigen. In other preferred embodiments, the nucleic acid further comprises a polyadenylation signal operably linked to the region encoding the tumor antigen. In still further embodiments, the nucleic acid further comprises an origin of replication. In defined embodiments, the nucleic acid may be a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus, pox-virus and adeno-associated virus or a bacterial vector or a plasmid vector. In other embodiments, the nucleic acid is packaged in a virus particle. In alternative preferred embodiments, the nucleic acid is packaged in a liposome.
The present invention provides a nucleic acid detection kit comprising, in suitable container means, an isolated nucleic acid segment comprising a contiguous nucleic acid sequence from SEQ ID NO:1, or a complement thereof. In preferred aspects, the kit further may comprise a detection reagent. In preferred embodiments, the detection reagent is a detectable label that is linked to the nucleic acid segment.
Other embodiments, contemplate a nucleic acid detection kit comprising, in suitable container means, an isolated nucleic acid segment that hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO:1, or a complement thereof. In particular aspects, the kit may comprise an isolated nucleic acid segment comprising a contiguous nucleic acid sequence from SEQ ID NO:1, or a complement thereof.
Particular aspects of the present invention contemplate a nucleic acid detection kit comprising, in suitable container means, a pair of primers for amplifying a nucleic acid having a sequence from SEQ ID NO:1, or a complement thereof. Other aspects provide a nucleic acid detection kit comprising, in suitable container means, a pair of primers with sequences from spatially distant regions of SEQ ID NO:1. In particular embodiments, the kit may comprise a pair of primers, each primer having a sequence from a spatially distant region of SEQ ID NO:1, or a complement thereof.
Also contemplated by the present invention is a composition comprising a purified HOJ-1 protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:2. Another aspect of the present invention provides a purified HOJ-1 protein having the amino acid sequence of SEQ ID NO:2. Yet another preferred embodiments provides a recombinant HOJ-1 protein or peptide prepared by expressing a DNA segment that encodes a HOJ-1 protein or peptide in a recombinant host cell and purifying the expressed HOJ-1 protein or peptide away from total recombinant host cell components.
The present invention further provides an isolated protein or polypeptide comprising a contiguous amino acid sequence encoded by a contiguous nucleic acid sequence from SEQ ID NO:1. Preferred embodiments provide an isolated protein or polypeptide having an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:1. In particular embodiments, the protein or polypeptide may comprise a contiguous amino acid sequence having the sequence of SEQ ID NO:2.
Other embodiments provide an isolated peptide, of between about 10 and about 50 amino acids in length, comprising a contiguous amino acid sequence encoded by a contiguous nucleic acid sequence from SEQ ID NO:1. In more particular embodiments the peptide is a HOJ-1 peptide that comprises the contiguous amino acid sequence of SEQ ID NO:2.
The present invention also contemplates an antibody that binds to a HOJ-1 protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:2. Other embodiments contemplate an antibody that binds to a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1. In preferred embodiments, the antibody binds to a HOJ-1 protein or peptide. In particular embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is operatively attached to a detectable label. In particular aspects, the label may be selected from the group consisting of a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme. In other embodiments, the antibody is operatively attached to a cytotoxic, anticellular or chemotherapeutic agent.
The present invention further provides a hybridoma cell that produces a monoclonal antibody that binds immunologically to a tumor antigen designated as HOJ-1. In preferred embodiments, the antibody does not bind immunologically to other human polypeptides. Other embodiments provide a polyclonal antisera, antibodies of which bind immunologically to a tumor antigen designated as HOJ-1.
Also provided by the present invention is an immunodetection kit comprising, in suitable container means, a first antibody that binds to a HOJ-1 protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:2. Other embodiments provide an immunodetection kit comprising, in suitable container means, a first antibody that binds to a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1 and an immunodetection reagent. In particular aspects, the kit comprises an antibody that binds to a HOJ-1 protein or peptide having the sequence as set forth in SEQ ID NO:2. In other aspects, the immunodetection reagent is a detectable label that is operatively attached to the first antibody. In still, further aspects, the immunodetection reagent is a detectable label that is operatively attached to a second antibody that has binding affinity for the first antibody. Other embodiments contemplate the kit of the present invention further comprising a second antibody that has binding affinity for the first antibody, and wherein the immunodetection reagent is a detectable label that is operatively attached to a third antibody that has binding affinity for the second antibody. In preferred embodiments, the first antibody is bound to a solid support. In other embodiments, the kit further comprises a suitably aliquoted composition of the protein or peptide to which the first antibody binds.
The present invention also provides a method for detecting a cancer cell, comprising identifying a cell that contains a HOJ-1 protein, peptide or mRNA. In preferred embodiments, the cancer cell is a metastatic cancer cell or a primary cancer cell. In further preferred embodiments, the cancer cell is a melanoma, glioblastomas, astrocytomas, leukemia, lymphoma, breast, gastric, colon, pancreas, renal, testicular, ovarian, lung, prostate, hepatic, germ cell or a lung cancer cell.
Other embodiments of the present invention provide a method for detecting a cancer cell, comprising identifying a cell that contains a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1. In preferred aspects the cancer cell is a metastatic cancer cell or a primary cancer cell. In other preferred aspects the cancer cell is a melanoma, glioblastomas, astrocytomas, leukemia, lymphoma, breast, gastric, colon, pancreas, renal, testicular, ovarian, lung, prostate, hepatic, germ cell or a lung cancer cell. In particularly preferred embodiments, the cell is identified by means of a molecular biological assay to identify a nucleic acid that encodes the protein or peptide. In certain aspects of the present invention the method comprises contacting sample nucleic acids from the cell with an isolated nucleic acid segment comprising a contiguous sequence SEQ ID NO:1, under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting the hybridized complementary nucleic acids thus formed. In preferred embodiments, the sample nucleic acids contacted are located within a cell. In other embodiments, the sample nucleic acids are separated from a cell prior to contact. In yet other embodiments, the sample nucleic acids are RNA. It is contemplated that the isolated nucleic acid segment comprises a detectable label and the hybridized complementary nucleic acids are detected by detecting the label. In further embodiments, the method may further comprise amplifying nucleic acids from the cell by conducting a polymerase chain reaction using a pair of primers, each primer having a sequence from a spatially distant region of SEQ ID NO:1, and detecting the amplified nucleic acids thus formed. Nucleic acids may also be amplified by nucleic acid amplification techniques other than the polymerase chain reaction.
In particularly defined embodiments, the method of detecting a cancer comprises the steps of contacting sample nucleic acids from the cell with a pair of nucleic acid primers that hybridize to distant sequences from SEQ ID NO:1, wherein the primers are capable of amplifying a corresponding nucleic acid segment when used in conjunction with a polymerase chain reaction; conducting a polymerase chain reaction to create amplification products or using other methods of nucleic acid amplification and detecting the amplification products xe2x80x9cthus formed. In particular embodiments, the amplification products thus formed are detected by contacting the amplification products with an isolated nucleic acid segment comprising a contiguous sequence from SEQ ID NO:1, under stringent hybridization conditions, and detecting amplification products that hybridize to the nucleic acid segment. In further embodiments, the amplification products may be formed using primers labeled with agents such as biotin, but not restricted to biotin, and the amplification products thus formed are detected directly by agents that recognize the primer label, often a fluorescently labelled agent that binds or recognizes the primer label.
In certain embodiments, it is contemplated that the cell is present within a biological sample obtained from a patient suspected of having cancer. In particular embodiments, the cell is identified by means of an immunoassay to identify the protein or peptide. In particularly preferred embodiments the cell is identified by contacting the cell or a sample therefrom with a first antibody that binds to a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1, under conditions effective to allow the formation of immune complexes, and detecting the immune complexes so formed. In further defined aspects, the first antibody is linked to a detectable label and the immune complexes are detected by detecting the presence of the label. In alternative preferred aspects the immune complexes are detected by means of a second antibody linked to a detectable label, the second antibody having binding affinity for the first antibody.
Also contemplated herein is a method of diagnosing a cancer comprising the steps of obtaining a sample from a subject; and determining the expression a HOJ-1 in cells of the sample. In particular aspects, the cancer may be selected from the group consisting of melanoma, leukemia, lymphoma, glioblastomas, astrocytomas, breast, gastric, colon, pancreas, renal, testicular, ovarian, lung, prostate, hepatic, lung cancer and germ cell tumors. In other aspects, the sample is a tissue or fluid sample. In preferred embodiments, the determining comprises assaying for a nucleic acid from the sample. In other aspects the method further comprises subjecting the sample to conditions suitable to amplify the nucleic acid. Preferred embodiments contemplate that the determining comprises contacting the sample with an antibody that binds immunologically to a HOJ-1 protein or peptide. In still further embodiments, the determining may further comprise subjecting proteins of the sample to ELISA. In particular aspects of the present invention, the diagnostic method further comprises the step of comparing the expression of HOJ-1 with the expression of HOJ-1 in non-cancer samples. In more particular aspects, the comparison involves evaluating the level of HOJ-1 expression. In further aspects, the comparison involves evaluating the structure of the HOJ-1 gene, protein or transcript.
The present invention provides a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a agent that inhibits HOJ-1 under conditions permitting the uptake of the agent by the tumor cell. In particular embodiments, the agent is encapsulated in a liposome. In other defined embodiments, the tumor cell is derived from a tissue selected from the group consisting of skin, brain, CNS, breast, gastric, colon, pancreas, renal, ovarian, lung, prostate, hepatic, lung tissue and germ cell tissue. In preferred aspects, the tissue may comprise melanoma, leukemia, lymphoma, glioblastomas, astrocytomas, breast, gastric, colon, pancreas, renal, testicular, ovarian, lung, prostate, hepatic, lung cancer and germ cell tumor cells. In particular aspects, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth and cell cycling.
A method for altering the phenotype of a tumor cell also is provided. The method comprises the step of contacting the cell with a nucleic acid (i) encoding antisense HOJ-1 and (ii) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the antisense HOJ-1, under conditions permitting the uptake of the nucleic acid by the tumor cell. In particular aspects of this embodiment, the tumor cell is derived from a tissue selected from the group consisting of skin, brain, CNS, breast, gastric, colon, pancreas, renal, ovarian, lung, prostate, hepatic, and lung tissue. In other aspects, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth or cell cycling. In preferred embodiments, the nucleic acid is encapsulated in a liposome or in a viral particle. In other embodiments, the nucleic acid is a viral vector selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, vaccinia virus, pox-virus and herpesvirus or bacterial vectors or plasmid vectors.
Also provided is a method for treating cancer, comprising inhibiting HOJ-1 within a patient with cancer. In particular aspects of this embodiments, the inhibiting comprises the step of contacting a tumor cell within a subject with a nucleic acid (i) encoding antisense HOJ-1 and (ii) a promoter active in the tumor cell, wherein the promoter is operably linked to the region encoding the antisense HOJ-1, under conditions permitting the uptake of the nucleic acid by the tumor cell. In other aspects, the inhibiting comprises administering to a patient with the cancer a biologically effective amount of composition comprising a HOJ-1 inhibitor. In preferred embodiments, the inhibitor comprises an antibody that binds to and inhibits the protein or peptide. In other preferred embodiments, the inhibitor comprises an immunotoxin that binds to and inhibits the protein or peptide. In still further embodiments, the inhibitor composition comprises a recombinant vector that expresses an agent that inhibits the production or activity of the protein or peptide. In preferred embodiments, the subject is a human.
Also provided is a method of predicting tumor metastasis comprising the steps of obtaining a sample from a subject; and determining the expression a HOJ-1 tumor antigen in cells of the sample. In particular aspects, the cancer is distinguished as metastatic and non-metastatic. In other aspects, the determining comprises assaying for a HOJ-1 nucleic acid or polypeptide in the sample.
The present invention also provides a method of screening a candidate substance for anti-tumor activity comprising the steps of providing a cell expressing HOJ-1 polypeptide; contacting the cell with the candidate substance; and determining the effect of the candidate substance on the cell. In preferred embodiments, the cell is a tumor cell. In other embodiments, the determining comprises comparing one or more characteristics of the cell in the presence of the candidate substance with characteristics of a cell in the absence of the candidate substance. In more particular embodiments, the characteristic is selected from the group consisting of HOJ-1 expression, MAGE-binding activity, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, chromosome recombination, tumor formation, tumor progression and tissue invasion. In defined embodiments, the candidate substance is a chemotherapeutic or radiotherapeutic agent. In other embodiments, the candidate substance is selected from a small molecule library. The cell may be contacted in vitro, in alternate embodiments, the cell is contacted in vivo.
The present invention provides a method of generating an anti-cancer immune response in a patient, comprising providing to a patient with the cancer an immunologically effective amount of a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1. In preferred aspects, the patient is provided with an immunologically effective amount of a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1. In other aspects, the patient is provided with an immunologically effective amount of a nucleic acid composition that encodes the protein or peptide.
Also contemplated is a therapeutic kit comprising, in suitable container means, an effective amount of a pharmaceutically acceptable formulation of an inhibitor that inhibits the production or activity of a protein or peptide encoded by a contiguous sequence from the nucleic acid sequence of SEQ ID NO:1. In preferred embodiments, the pharmaceutical formulation is suitable for parenteral administration.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.